Conjugates for targeting and clearing aggregates

ABSTRACT

In some embodiments, the disclosure provides for a conjugate comprising: a) a peptide that competes with an Fc fragment of an IgG for binding to an Fc receptor; and b) a targeting moiety that targets molecular aggregates. In some embodiments, the disclosure provides for methods of using the conjugates for treating a disease or disorder associated with aggregate formation.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/664,345, filed on Apr. 30, 2018. The foregoingapplication is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 26, 2019, isnamed 1848081-0002-107_101_SL.txt and is 7,036 bytes in size.

BACKGROUND OF THE INVENTION

Numerous diseases and disorders are caused by or associated withaggregates of proteins, polysaccharides, lipids and/or nucleotides. Inmany cases, these aggregates are considered to be toxic. These diseasesaffect various cells, tissues and organs throughout the body.

Amyloidosis is a category of diseases associated with accumulation ofabnormal protein conformers, known as amyloid fibrils, in tissues.Clinical features depend on the type of amyloidosis, and may includediarrhea, weight loss, fatigue, glossomegaly, haemorrhage, somaticsensory deficits, postural hypotension, peripheral edema andsplenomegaly. Treatment of amyloidosis typically addresses symptomsonly, and may not be effective. Examples of amyloidosis include ALamyloidosis, AA amyloidosis, transthyretin amyloidosis, leptomeningealamyloidosis, type II diabetes, and certain neurodegenerative diseasessuch as Alzheimer's Disease and Creutzfeldt-Jakob Disease.

Glycogen storage diseases are a series of diseases typically associatedwith enzyme deficiencies resulting in aberrant glycogen synthesis orglycolysis. These diseases may be genetic or acquired and symptoms varydepending on the type of disease. Examples of glycogen storage diseasesinclude GSDI-XV. Treatment is typically a diet of limited carbohydrates,but may include treatment with allopurinol and/or human granulocytecolony stimulating factor.

Lipid aggregate diseases include atherosclerosis, heart disease, andneurodegenerative diseases such as Niemann-Pick Type C disease. Diseasesin which abnormally aggregating RNA molecules have been implicatedinclude Dentatorubral pallidoluysian atrophy (DRPLA), Huntington chorea(HD), Oculopharyngeal muscular dystrophy (OPMD), Spinobulbar muscularatrophy (SBMA), and Spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17(SCA1, SCA2, SCA3, SCA6, SCAT, SCA1 7), Fragile XA, Fragile XE,Friedrich's ataxia, Myotonic Dystrophy type 1 (DM1), Myotonic Dystrophytype 2 (DM2), Spinocerebellar ataxia types 8, 10, and 12 (SCAB, SCA 10,SCA 12), amyotrophic lateral sclerosis and fronotemporal dementia (FTD).These diseases lack an effective treatment capable of clearing theaggregates.

Degenerative diseases of the nervous system impose a significantworldwide medical and public health burden. The prevalence and incidenceof these diseases rise dramatically with age and the number of cases isexpected to increase with extended life expectancy in many countries(Checkoway H et al. IARC Sci Publ. 2011; (163):407-19). Alzheimer'sDisease International (ADI) estimated in 2015 that there were 46.8million people with Alzheimer's Disease worldwide, and that this numberwill grow to 131.5 million people by 2050 (Prince M. et al. ADI Report.2016 Sep. 20; 1-131). An estimated 5.7 million Americans of all ageshave Alzheimer's disease in 2018, of which 5.5 million people are age 65and older (Alzheimer's Association. 2018 Alzheimer's Disease Facts andFigures. Alzheimers Dement 2018; 14(3):367-429). By 2050, the number ofpeople age 65 and older with Alzheimer's disease in the U.S. may nearlytriple, from 5.1 million to a projected 14 million, in the absence ofmedical intervention to prevent or cure the disease. Id.

Neurodegenerative diseases are commonly associated with the accumulationof intracellular or extracellular protein aggregates, such asα-synuclein in Parkinson's disease, β-amyloid and tau in Alzheimer'sdisease, huntingtin in Huntington's disease and prion protein (PrP) intransmissible prion encephalopathies (Brundin P et al. Nat Rev Mol CellBiol. 2010 April; 11(4): 301-307). Other systemic degenerative diseases,some of which occur outside the nervous system, are also attributed toα-synuclein, β-amyloid, huntingtin, prion protein, and amylin. Theseinclude diseases such as type II diabetes, characterized by amylindeposition, and amyloidosis, a rare disease in which amyloid proteinbuilds up in various organs and tissues such as the heart, kidney,liver, spleen, nervous system, or digestive tract.

A new understanding is emerging about protein aggregate disorders suchas Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,amyotrophic lateral sclerosis (ALS), transmissible spongiformencephalopathies (TSEs), motor neuron diseases, tauopathies, type IIdiabetes, amyloidosis etc that involve protein misfolding. Evidencesuggests that protein-misfolding and subsequent propagation of theserogue proteins is a generic phenomenon shared with diseases caused bytau, α-synucleins and β-amyloid proteins (Panegyres P K and Armari E. AmJ Neurodegener Dis. 2013; 2(3): 176-186). The deposition of aggregatedprotein is a central feature of these otherwise unrelated pathologicalconditions.

Protein aggregation of α-synuclein, β-amyloid, huntingtin, prion proteintypically begins when several identical monomeric proteinsself-associate to change conformation from native structure toaggregated structures. It is believed that in the majority ofaggregation diseases, a conformational transition from native structureto β-sheet-rich oligomeric structures is critical even in thefibrillation process (Morales B et al. CNS Neurol Disord Drug Targets.2009 November; 8(5): 363-371). Thus, numerous therapies attempt to blockpropagation of protein misfolding throughout the brain and body (Frost Band Diamond M I. Nat Rev Neurosci. 2010 March; 11(3): 155-159). Some ofthese strategies include (1) inhibition of the production of amyloidpolypeptides, (2) inhibition of the formation of amyloid fibrils, and(3) destabilization of amyloid structures using compounds, antibodies,vaccines, small molecules, antioxidants, destabilizing peptides, ornatural extracts.

There is currently no cure for aggregate diseases such as Alzheimer'sDisease, and the prognosis in Alzheimer's Disease is poor. As of 2018,the majority of treatments are limited to symptomatic management whichconsists primarily of acetylcholinesterase inhibitors. There are onlyfive approved medications in the US for treating Alzheimer's Disease(Aricept®, Exelon®, Razadyne®, Namenda®, and Donepezil®). Thesemedications temporarily slow the worsening of symptoms and improvequality of life for those with Alzheimer's and their caregivers.However, they do not cure Alzheimer's or stop it from progressing.

In view of the lack of effective therapeutic options foraggregate-associated diseases or disorders, there is a need forinnovative therapeutics for treating these diseases or disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a schematic of the synthesis of BDB, a conjugate comprisinga specific carboxylic acid derivative of thioflavin T (Compound 1) and aspecific targeting moiety (cp33 peptide derivative; SEQ ID NO:3). Asshown, Compound 1 is added as the terminal residue of the biotinylatedsynthetic cyclic peptide, which is a derivative of the cp33 peptide(Bonetto S et al. FASEB J. 2009; 23(2):575-85), via a linker. In someembodiments, linkers may comprise 13-alanine, 9-amino-4,7-dioxanonanoicacid and/or 6-amino-4-oxaohexanoic acid.

FIG. 2 shows data demonstrating BDB binding to various amyloids. Asshown, BDB and Tht have increased fluorescence in the presence ofamyloid proteins, as compared to amyloid-free control reactions. Thecp33 peptide not conjugated with Compound 1 did not show a fluorescencesignal in the presence of amyloid, as compared to amyloid-free controlreactions.

FIG. 3 shows data for BDB binding to activated phagocytic cells fromhumans and mice. As shown, U937 cells treated with IFNγ (IFNg) or murineprimary macrophages treated with LPS, showed enhanced binding of BDB inflow-cytometry experiments. These results are consistent with BDBbinding to the increased surface expression of Fc receptors. IgG1 bindsmultiple receptors with high affinity and labels cells irrespective ofactivation status.

FIG. 4 shows the binding curve for BDB to IFNγ-activated U937 cells. Asshown, increasing concentrations of BDB resulted in as many as 80% ofIFNγ-activated U937 cells being bound, in flow-cytometry experiments.Half-maximal binding was see at approximately 200 nM BDB. IgG1 bound toall cells under the same conditions.

FIG. 5 shows data from a competition assay between human IgG1, BDB, andcp33 peptide for binding to IFNγ-activated U937 cells, in flow-cytometryexperiments. As shown, when the concentration of human IgG1 increasesabove approximately 10 nM, both the BDB and cp33 peptide are displacedfrom binding to IFNγ-activated U937 cells. This competition isconsistent with the BDB and cp33 peptides binding to the Fc receptor.

FIG. 6 shows data comparing the localization of amyloid to the surfaceof IFNγ-activated U937 cells treated with BDB, cp33 peptide, or vehiclecontrol, in flow-cytometry experiments. As shown, BDB enhances thebinding of amyloid to IFNγ-activated U937 cells. Both BDB and cp33peptide show cell binding irrespective of whether amyloid is present(upward displacement of the cell population on the y-axis in top fourpanels). Up to 14.4% of IFNγ-activated U937 cells are observed bindingamyloid in the presence of BDB as compared to 1.0% in the presence ofcp33 peptide or 0.4% when treated with vehicle.

FIG. 7 shows labelled amyloid phagocytosis data formacrophage-differentiated U937 cells co-incubated with pHrodo-redlabelled amyloid, BDB, cp33 peptide, or control. As shown, BDB enhancesthe phagocytosis of amyloid by macrophage-differentiated U937 cells. Redcolour (y-axis) reported phagocytosed amyloid; this signal increasedmore rapidly and achieved a greater amplitude in cells treated withBDB-amyloid (1) as compared to cp33-amyloid (3) or amyloid alone (2).pHrodo-red E. coli were added as a positive control (4). In the absenceof amyloid, BDB (5) and the cp33 peptide (6) did not generate a signal.

FIG. 8 shows labelled amyloid phagocytosis data formacrophage-differentiated U937 cells co-incubated with pHrodo-redlabelled lysozyme amyloid, cp33 or various BDB constructs. Cp33=peptidealone (no dye); scBDB1=BDB1 with scrambled peptide portion;BDB1=linkerless BDB construct; BDB2=BDB construct comprising thebeta-alanine linker; BDB3=BDB construct comprising the6-amino-4-oxahexanoate linker; and BDB4=BDB construct comprising the9-amino-4,7-dioxanonanoate linker. Phagocytosis was plotted in arbitraryfluorescence units at the 2 hour timepoint using the automated analysisof pHrodo-red pixels with an Incucyte microscope.

FIG. 9 shows space-filing models of how the amyloid-binding domain(pink) may be separated from the FcR-binding domain (grey) by linkers ofdiffering length (green). The structural effects of adding linkersbetween the amyloid binding and FcR-binding moieties is demonstrated. Insome embodiments, linkers may comprise β-alanine;9-amino-4,7-dioxanonanoic acid (short PEG linker) and6-amino-4-oxaohexanoic acid (long PEG linker).

SUMMARY OF THE INVENTION

In some embodiments, the disclosure provides for a conjugate comprising:a) a peptide that competes with an Fc fragment of an IgG for binding toan Fc receptor; and b) a targeting moiety that targets molecularaggregates. In some embodiments, the peptide activates Fc effectorfunction. In some embodiments, the Fc receptor is selected from thegroup consisting of: FcγRI, FcγRII, and FcγRIII. In some embodiments,the Fc receptor is a human receptor. In some embodiments, the Fcreceptor is any one of a FcγRI, FcRn, TREM2, CD33, TMEM119, CD11b, CD45,Iba1, CX3CR1, CD68, P2X family or P2Y family receptor. In someembodiments, the Fc receptor is TREM2. In some embodiments, the Fcreceptor is FcRn. In some embodiments, the Fc receptor is human FcγRI(hFcγRI). In some embodiments, the Fc effector function is phagocytosis.In some embodiments, the peptide competes with the Fc portion of an IgG3or IgG1 antibody. In some embodiments, the peptide competes with the Fcportion of IgG1. In some embodiments, the peptide comprises a tripeptidemotif of LLG. In some embodiments, the peptide comprises a dimer ofmotif ζP and wherein ζ represents a hydrophobic residue. In someembodiments, the peptide comprises threonine at an N-terminus of thepeptide. In some embodiments, the peptide comprises glutamic acid at aC-terminus of the peptide. In some embodiments, the peptide comprises anamino acid sequence of TX2CXXζPXLLGCφXE (SEQ ID NO: 1); wherein X is anyamino acid, ζ is a hydrophobic residue and φ is an acidic amino acid. Insome embodiments, the peptide binds specifically to hFcγRI and does notexhibit cross-reactivity with hFcγRII or hFcγRIII. In some embodiments,the peptide comprises the amino acid sequence of AQVNSCLLLPNLLGC (SEQ IDNO: 2). In some embodiments, the peptide comprises the amino acidsequence of AQVNSCLLLPNLLGCGDDK (SEQ ID NO: 3). In some embodiments, oneor more amino acid residues in the peptide are methylated. In someembodiments, the asparagine residue at the amino acid positioncorresponding to position 11 of SEQ ID NO: 2 or 3 is methylated. In someembodiments, the leucine residue at the amino acid positioncorresponding to position 13 of SEQ ID NO: 2 or 3 is methylated.

In some embodiments, the disclosure provides for a conjugate multimercomprising two or more of any of the conjugates disclosed herein, linkedto each other to generate a dimeric or oligomeric peptide. In someembodiments, the peptide binds to two hFcγRIs and activates Fc effectorfunction. In some embodiments, the conjugate comprises two Fc mimeticpeptides linked by a KKKKK linker (SEQ ID NO: 22). In some embodiments,the conjugate is capable of recruiting an immune cell to the molecularaggregates. In some embodiments, the immune cell is a monocyte-derivedcell. In some embodiments, the immune cell is a macrophage. In someembodiments, the immune cell is a monocyte. In some embodiments, theimmune cell is a microglial cell. In some embodiments, the conjugatebinds to the immune cell and facilitates activation of the immune cell.In some embodiments, the conjugate facilitates phagocytosis of at leasta portion of the molecular aggregates. In some embodiments, thetargeting moiety targets protein aggregates. In some embodiments, theprotein aggregates comprise aggregates of any one of the followingproteins: alpha-synuclein, tau, amyloid beta, TDP-43, SOD1, FUS, TDP-43,prion protein, immunoglobulin light chain, transthyretin, fibrinogen,collagen, islet amyloid polypeptide, lysozyme, calcitonin, serum amyloidA protein, LECT2, amylin, gelsolin, fibrinogen A, prolactin,keratoepithelin, and β₂-microglobin. In some embodiments, the targetingmoiety targets lipid aggregates. In some embodiments, the lipidaggregates are cholesterol aggregates. In some embodiments, thetargeting moiety targets polysaccharide aggregates. In some embodiments,the targeting moiety targets glycogen aggregates. In some embodiments,the targeting moiety is a small molecule. In some embodiments, thetargeting moiety is a peptide. In some embodiments, the targeting moietyis a nucleic acid. In some embodiments, the targeting moiety is a dye.In some embodiments, the dye binds to amyloid. In some embodiments, thedye is selected from the group consisting of: a thioflavin, pentamerformyl thiophene acetic acid (pFTAA), heptamer formyl thiophene aceticacid (hFTAA), Congo Red, crystal violet, 1-amino-8-naphtalene sulphonate(ANS), 4-(dicyanovinyl)-julolidine (DCVJ), aminobenzanthrone, MethoxyXO4, thiazin red, Pittsburgh B, and amyloid-binding derivatives thereof.In some embodiments, the thioflavin is thioflavin T, or anamyloid-binding derivative thereof. In some embodiments, the amyloidcomprises any one or more of the following proteins: alpha-synuclein,tau, amyloid beta, TDP-43, SOD1, FUS, C79ORF, TDP-43, prion protein,immunoglobulin light chain, transthyretin, fibrinogen, collagen, isletamyloid polypeptide, lysozyme, calcitonin, serum amyloid A protein,LECT2, amylin, gelsolin, fibrinogen A, prolactin, keratoepithelin, andβ₂-microglobin. In some embodiments, the amyloid comprises lysozyme. Insome embodiments, the dye binds to cholesterol. In some embodiments, thedye is filipin, or a cholesterol-binding derivative thereof. In someembodiments, the dye binds to collagen. In some embodiments, the dye issirius red, Col-F, Oregon Green 488, or Picrosirius red, Light Green SFyellowish, Fast Green FCF, methyl blue, water blue, aniline blue, andcollagen-binding derivatives thereof. In some embodiments, the dye bindspolysaccharide aggregates. In some embodiments, the dye binds glycogenaggregates. In some embodiments, the dye is selected from the groupconsisting of periodic acid, alcian blue, and Dimethyl methylene blue.In some embodiments, the dye binds nucleotide aggregates. In someembodiments, the dye binds to RNA aggregates. In some embodiments, thedye binds DNA aggregates. In some embodiments, the targeting moiety hasbeen modified to facilitate conjugation to the peptide. In someembodiments, the targeting moiety has been modified such that itincludes a carboxyl group. In some embodiments, the carboxyl group ofthe targeting moiety is bonded to the amino terminus of the peptide. Insome embodiments, the targeting moiety has been modified such that itincludes an amino group. In some embodiments, the amino group of thetargeting moiety is bonded to the carboxy terminus of the peptide.

In some embodiments, the targeting moiety is conjugated to the peptideby means of a linker. In some embodiments, the linker is a non-cleavablelinker, hydrazone linker, thioether linker, disulfide linker, peptidelinker or β-glucuronide linker. In some embodiments, the linker is arigid linker. In some embodiments, the linker comprises the structure ofCompound II.

wherein n is 1-30, and m is 0, 1, 2, 3, 4, or 5. In some embodiments, nis 1-25, 1-20, 1-15, 1-10, 1-8, 1-6, 1-4, 1-2 or 1. In some embodiments,n is 2. In some embodiments, m is 0. In some embodiments, the linker isa β-alanine linker, a short polyethylene glycol (PEG) linker, or a longPEG linker. In some embodiments, the linker is a β-alanine linker.

In some embodiments, the conjugate is further conjugated to anadditional heterologous moiety. In some embodiments, the heterologousmoiety is biotin. In some embodiments, the conjugate comprises the aminoacid sequence of AQVNSCLLLPNLLGCGDDK (SEQ ID NO: 3) fused to thioflavinT, or a derivative thereof. In some embodiments, the conjugate comprisesthe amino acid sequence of AQVNSCLLLPNLLGCGDDK (SEQ ID NO: 3) fused toCompound I:

In some embodiments, the conjugate comprises the structure of CompoundIII:

wherein R¹ is biotin or hydrogen;wherein R² comprises:

In some embodiments, R¹ is biotin. In some embodiments, R¹ is hydrogen.In some embodiments, R² comprises

In some embodiments, the disclosure provides for a method of treating anamyloidosis by administering any one or more of the conjugates disclosedherein. In some embodiments, the amyloidosis is selected from the groupconsisting of: AL amyloidosis, AA amyloidosis, Alzheimer Disease, LECT2amyloidosis, leptomeningeal amyloidosis, senile systemic amyloidosis,familial amyloid polyneuropathy, haemodialysis-associated amyloidosis,type 2 diabetes, Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, Finnish type amyloidosis,cerebral amyloid angiopathy, familial visceral amyloidosis, primarycutaneous amyloidosis prolactinoma, familial corneal amyloidosis,Parkinson's Disease, amyotrophic lateral sclerosis, cerebral amyloidangiopathy, frontotemporal lobar dementia, medullary thyroid carcinoma,and B2M amyloidosis. In some embodiments, the amyloidosis is a familialamyloidosis. In some embodiments, the amyloidosis is systemicamyloidosis.

In some embodiments, the disclosure provides for a method of reducinglevels of an aggregate in a cell or tissue by treating the cell ortissue with any of the conjugates disclosed herein. In some embodiments,the disclosure provides for a composition comprising any of theconjugates disclosed herein and a pharmaceutically acceptable excipient.

DETAILED DESCRIPTION

The terms “linker” and “linkage” are used interchangeably and mean achemical moiety comprising a covalent bond or a chain of atoms thatcovalently attaches, or is attached to, two different entities (e.g.,any of the targeting moieties disclosed herein and any of the peptidesdisclosed herein).

“Linking moiety” means a chemically reactive group, substituent ormoiety, e.g. a nucleophile or electrophile, capable of reacting withanother molecule to form a linkage by a covalent bond.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

“Percent (%) sequence identity” or “percent (%) identical” with respectto a reference peptide (or nucleotide) sequence is defined as thepercentage of amino acid residues (or nucleic acids) in a candidatesequence that are identical to the amino acid residues (or nucleicacids) in the reference peptide (nucleotide) sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acid(nucleic acid) sequence identity values are generated using the sequencecomparison computer program ALIGN-2. The ALIGN-2 sequence comparisoncomputer program was authored by Genentech, Inc., and the source codehas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablefrom Genentech, Inc., South San Francisco, Calif., or may be compiledfrom the source code. The ALIGN-2 program should be compiled for use ona UNIX operating system, including digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges, as well as any individual numbers or ranges present withinthe disclosed numeric range. For example, a disclosed range of “1-10”would include ranges such as “1-5”, “2-6”, “7-9”, and “5-10”

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers. As used herein, the term “comprises”also encompasses the use of the narrower terms “consisting” and“consisting essentially of.”

The term “consisting essentially of” is limited to the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristics of the invention(s) disclosed herein.

“Amino acid” includes both natural amino acid and substituted aminoacids. “Natural amino acid” refers to any of the commonly occurringamino acids as generally accepted in the peptide art and representL-amino acids unless otherwise designated (with the exception of achiralamino acids such as glycine), including the canonical 20 amino acidsencoded directly by the genetic code, as well as selenocysteine,selenomethionine, and ornithine. “Substituted amino acid” refers to anamino acid containing one or more additional chemical moieties that arenot normally a part of the amino acid. Such substitutions can beintroduced by a targeted derivatizing agent that is capable of reactingwith selected side chains or terminal residues and via otherart-accepted methods. For example, cysteinyl residues most commonly arereacted with alpha-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues can also bederivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidazoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole. In some embodiments, carboxylside groups (aspartyl or glutamyl) can be selectively modified byreaction with carbodiimides such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4, 4-dimethylpentyl) carbodiimide. In some embodiments, aspartyland glutamyl residues can be converted to asparaginyl and glutaminylresidues by reaction with ammonium ions. Glutaminyl and asparaginylresidues may be deamidated to the corresponding glutamyl and aspartylresidues. Alternatively, residues may be deamidated under mildly acidicconditions. Other modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the alpha-amino groups of lysine, arginine and histidineside chains (see, e.g., T. E. Creighton, Proteins: Structure andMolecule Properties, W. H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation ofC-terminal carboxyl groups. Blocking groups and/or activating groups mayalso be incorporated.

The terms “peptide”, “polypeptide”, “oligopeptide”, and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. The chain may be linear, branched, or cyclic, it may comprisemodified amino acids, and/or may be interrupted by non-amino acids. Theterms also encompass an amino acid chain that has been modifiednaturally or by intervention; for example, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labellingcomponent. Also included within the definition are, for example,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), as well as othermodifications known in the art. It is understood that the peptides canoccur as single chains or associated chains.

1. The Peptides

In some embodiments, the conjugates comprise a peptide capable ofactivating an immune cell. In some embodiments, the peptide is capableof recruiting an immune cell. In some embodiments, the peptide iscapable of competing with an Fc fragment of an IgG for binding to an Fcreceptor. In some embodiments, the peptide is any of the peptidesdescribed in WO 2009/090268, which is incorporated by reference hereinin its entirety.

In some embodiments, the peptide is less than 50, 45, 40, 35, 30, 25,20, 15, or 10 amino acids in length. In some embodiments, the peptide isless than 25 amino acids in length. In some embodiments, the peptide isless than 20 amino acids in length. In some embodiments, the peptide isbetween 5-50, 10-50, 5-40, 10-40, 5-30, 10-30, 5-25, 10-25, 5-20, 10-20,or 15-25 amino acids in length.

The term peptide includes peptides containing post-translationalmodifications of the peptide, for example, glycosylations, acetylations,phosphorylations, and sulphations. In addition, protein fragments,analogs (including amino acids not encoded by the genetic code, e.g.homocysteine, ornithine, D-amino acids, and creatine), natural orartificial mutants or variants or combinations thereof, fusion proteins,derivatized residues (e.g. alkylation of amine groups, acetylations oresterifications of carboxyl groups) and the like are included within themeaning of peptide. By “modified” with reference to peptides, is meant amodification in one or more functional groups, for example any portionof an amino acid, the structure and/or location of a sugar or othercarbohydrate, or other substituents of biomolecules, and can includewithout limitation chemical modifications (e.g., succinylation,acylation, the structure and/or location of disulfide bonds), as well asnoncovalent binding (e.g., of a small molecule, including a drug).

A peptide according to an embodiment of the present invention may beused herein to refer to constrained (i.e. having some element allowingcyclisation between two backbone termini, two side chains, or one of thetermini and a side chain, as for example, amide or disulfide bonds) orunconstrained (e.g. linear) amino acid sequences of less than about 50amino acid residues, such as less than about 40, 30, 20 or 10 amino acidresidues. This list may also include oligomers, such as 3, 4 or 5peptides linked together or dimers comprising 2 peptides linked togetherby means of a peptide linker, for example. In particular embodiments,the peptides are between about 10 and about 30 amino acid residues, andin more particular embodiments, 16 to 18 amino acid residues. However,on reading the present disclosure, it will be apparent to the skilledperson that it is not the length of a particular peptide that isrequired but its ability to bind to an Fc receptor (e.g., FcγRI) andcompete with the binding of IgG. For example, amino acid sequences of 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24and 25 amino acid residues are contemplated to be peptide compoundswithin the context of the present invention. A dimeric peptide may havean amino acid sequence comprising two peptide amino acid sequenceslinked by a linker. The linker may comprise an amino acid sequence ofless than about 10 amino acid residues, such as 9, 8, 7, 6, 5, 4 or 3amino acid residues. The linker amino acid residues may be of a singleamino acid or combinations of different amino acids. For example,combinations of glycine (G) and serine (S) may be used. Alternatively,the linker may comprise residues of glycine, serine or lysine (K) only.

A peptide of the invention that is able to mimic biological activity ofan Fc fragment, such as effector function may be termed a ‘Fc mimeticpeptide’. Effector function of an Fc fragment as encompassed by, but notlimited by, the present invention comprises phagocytosis, CDC and/orADCC. In some embodiments, the biologically active peptide binds to anFc receptor, e.g., a Fcγ receptor. In some embodiments, such a peptidecompetes with the Fc fragment of IgG for binding to Fc receptor (e.g., aFcγ receptor) and therefore binds to the same or an overlapping bindingsite on the Fc receptor (e.g., Fcγ receptor) as the Fc fragment. Presentin a complex form (multimeric), which may include dimers and oligomers,an Fc mimetic peptide may trigger effector functions. Present in solubleform (monomeric), an Fc mimetic peptide can inhibit effector functions.In some embodiments, these peptides in soluble form may be able toactivate effector functions if the IC₅₀ concentration of the solubleform required to inhibit the effector function is lower than 10-20 μM.

A peptide that is unable to mimic the effector functions of an Fcfragment of an IgG to an Fcγ receptor, namely to trigger effectorfunctions in multimeric form and inhibit effector functions in solubleform may be referred to as ‘Fc an non-mimetic peptide’. Using thisterminology, four groups of peptides may be defined: i) the first groupcomprises a peptide that competes with the Fc fragment for binding tothe Fc receptor (e.g., Fcγ receptor) and therefore binds to the samebinding site on the Fc receptor (e.g., Fcγ receptor) as the Fc fragment.However, such peptides cannot activate effector functions in a complexedform but can activate effector functions when in soluble form. In someembodiments, these peptides may have lost their ability to triggereffector functions if the IC₅₀ concentration of the soluble formrequired to inhibit the effector function is higher than 10-20 μM andhence the avidity of the complexed peptide is sufficiently low. ii) Thesecond group comprises a peptide that competes with the Fc fragment ofan IgG for binding to the Fc receptor (e.g., Fcγ receptor) and thereforebinds to the same binding site on the Fc receptor (e.g., Fcγ receptor)as the Fc fragment. In some embodiments, this peptide can activateeffector functions when in a dimeric as well as when in a complexed formi.e. as a soluble cyclic peptide dimer. Such peptides may also be knownas committed agonists. iii) The third group comprises a peptide thatdoes not compete with the Fc fragment of an IgG for binding to the Fcreceptor (e.g., Fcγ receptor). These peptides however can activate theFc receptor (e.g., Fcγ receptor) when in complexed form (Berntzen et al,2006). iv) The fourth group comprises a peptide that does not competewith the Fc fragment of an IgG for binding to the Fc receptor (e.g., Fcγreceptor) but is able to bind to the Fc receptor (e.g., Fcγ receptor) ata site distinct from the Fc binding site. These peptides cannot activateor inhibit effector functions in complexed or soluble form. Thesepeptides may have an affinity and avidity that is too high to activateeffector function.

In some embodiments, any of the peptides disclosed herein may compriseboth naturally and non-naturally occurring amino acid sequences. Bynon-naturally occurring is meant that the amino acid sequence is notfound in nature. In some embodiments, non-naturally occurring amino acidsequences have between about 10 and 30 amino acid residues,alternatively about 20 amino acid residues. These include peptides,peptide analogs, peptoid and peptidomimetics containing naturally aswell as non-naturally occurring amino acids. In a specific aspect, thepeptides of the invention comprise amino acid residues consisting ofonly naturally occurring amino acids.

A C-terminal region of an immunoglobulin heavy chain that also comprisesthe hinge region between the two constant domains CH1 and CH2 may bereferred to as a ‘Fc fragment.’ This fragment of the C-terminal regionmay be a native sequence Fc fragment or a variant Fc fragment. Althoughthe boundaries of the Fc fragment of an immunoglobulin heavy chain canvary, the human IgG heavy chain Fc fragment is usually defined tostretch from an amino acid residue at position 231 to thecarboxyl-terminus thereof. With the upper and core hinge, the ‘Fcfragment’ starts from position 216 (EU nomenclature according to Rabat(1987, 1991)). The Fc fragment of an immunoglobulin generally comprisestwo constant domains, CH2 and CH3. The CH2 domain of a human IgG Fcfragment usually extends from about amino acid 231 to about amino acid340. The CH3 domain of a human IgG Fc fragment usually extends fromabout amino acid 341 to about amino acid residue 447 of a human IgG(i.e. comprises the residues C-terminal to a CH2 domain). In someembodiments of the present invention, the variant IgG Fc fragment may beselected from IgG1, IgG2, IgG3 or IgG4. In particular embodiments, theIgG Fc fragment of IgG1. In some embodiments, the IgG1 Fc may be writtenin the alternative as Fcγ1. A ‘hinge fragment’ is generally defined asstretching from Glu 216 to Pro 230 of human IgG1, or the equivalentpositions in IgG2, IgG3 or IgG4 (Burton, 1985). A functional Fc fragmentpossesses an effector function of a native sequence Fc fragment forexample: C1q binding, CDC, Fc receptor binding, phagocytosis,endocytosis of opsonized particles, antigen presentation, release ofinflammatory mediators (e.g. IL-6, TNFα, IL-1), cellular cooperation,superoxide burst, ADCC, down regulation of cell surface receptors (e.g.B cell receptor), etc. Effector function of an Fc fragment asencompassed by, but not limited by the present invention comprisesphagocytosis, CDC and/or ADCC.

As is generally known in the art, an Fcγ receptor (FcγR) is a receptorthat binds an IgG antibody and includes receptors of the FcγRI, FcγRIIand FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIa (anactivating receptor) and FcγRIIb (an inhibiting receptor), which havesimilar amino acid sequences that differ primarily in the cytoplasmicdomains thereof. Fc receptors are reviewed in Ravetch and Kinet (1991,Annu. Rev. Immunol 9: 457-92); Capel et al., (1994, Immunomethods 4:25-34); and de Haas et al., (1995, J. Lab. Clin. Med. 126: 330-41).

A peptide according to an embodiment of the present invention may beobtained from a library of peptides that are able to bind to an Fcγreceptor such as FcγRI. The library may be displayed on particles ormolecular complexes, e.g. replicable genetic packages, such as yeast,bacterial or bacteriophage (e.g. T7) particles, viruses, cells orcovalent, ribosomal, microbead or other in vitro display systems, eachparticle or molecular complex containing nucleic acid encoding thepeptide. Phage display is described in WO92/01047 and e.g. U.S. Pat.Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215,5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650,6,492,160 and 6,521,404, each of which is herein incorporated byreference in their entirety.

Following selection of peptides of the invention able to bind an FcγRIreceptor and displayed on bacteriophage or other library particles ormolecular complexes, nucleic acid may be taken from a bacteriophage orother particle or molecular complex displaying a said selected peptide.Such nucleic acid may be used in subsequent production of a peptide byexpression from nucleic acid with the sequence of nucleic acid takenfrom a bacteriophage or other particle or molecular complex displaying asaid peptide.

In some embodiments, a peptide of the invention in soluble form can bindto the FcγRI without eliciting an effector response. In someembodiments, in order to activate the receptor it is necessary forreceptor clustering to occur.

In some embodiments, ability to bind FcγRI may be further tested, alsoability to compete with e.g. an Fc fragment of an IgG for binding toFcγRI. In some embodiments, a peptide according to the present inventionmay bind FcγRI with the affinity of a functional Fc fragment or with anaffinity that is better or lower, as measured by, for example, BIACORE.

Binding affinity of different peptides can be compared under appropriateconditions.

The techniques required to make substitutions within amino acidsequences of peptides of the invention are available in the art. Variantsequences may be made, with substitutions that may or may not bepredicted to have a minimal or beneficial effect on effector function,and tested for ability to bind Fc receptors and/or for any other desiredproperty.

In some embodiments, the peptide comprises a sequence that has at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%amino acid sequence homology with a sequence of any one of SEQ ID Nos:1-21, or a functional fragment thereof. In some embodiments, the peptidecomprises a sequence that has at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identitywith a sequence of any one of SEQ ID Nos: 1-21, or a functional fragmentthereof. In the context of any of the peptides disclosed herein, a“functional fragment thereof” is defined as being capable of binding toand/or activating an Fc receptor (e.g., hFcγRI).

In some embodiments, the peptide comprises an amino acid sequence ofTX2CXXζPXLLGCφXE (SEQ ID NO: 1); wherein X is any amino acid, ζ is ahydrophobic residue and φ is an acidic amino acid. In some embodiments,the peptide binds specifically to hFcγRI and does not exhibitcross-reactivity with hFcγRII or hFcγRIII. In some embodiments, thepeptide comprises the amino acid sequence of AQVNSCLLLPNLLGC (SEQ ID NO:2). In some embodiments, the peptide comprises the amino acid sequenceof AQVNSCLLLPNLLGCGDDK (SEQ ID NO: 3). In some embodiments, the peptidecomprises the amino acid sequence of any one of:

(SEQ ID NO: 4) TDTCLMLPLLLGCDEE (SEQ ID NO: 5) DPICWYFPRLLGCTTL(SEQ ID NO: 6) WYPCYIYPRLLGCDGD (SEQ ID NO: 7) GNICMLIPGLLGCSYE(SEQ ID NO: 8) VNSCLLLPNLLGCGDD (SEQ ID NO: 9) TPVCILLPSLLGCDTQ(SEQ ID NO: 10) TVLCSLWPELLGCPPE (SEQ ID NO: 11) TFSCLMWPWLLGCESL(SEQ ID NO: 12) FGTCYTWPWLLGCEGF (SEQ ID NO: 13) SLFCRLLLTPVGCVSQ(SEQ ID NO: 14) HLLVLPRGLLGCTTLA (SEQ ID NO: 15) TSLCSMFPDLLGCFNL(SEQ ID NO: 16) SHPCGRLPMLLGCAES (SEQ ID NO: 17) TSTCSMVPGPLGAVSTW(SEQ ID NO: 18) KDPCTRWAMLLGCDGE (SEQ ID NO: 19) IMTCSVYPFLLGCVDK(SEQ ID NO: 20) IHSCAHVMRLLGCWSR (SEQ ID NO: 21)AQVNSCLLLPNLLGCSYEKKKKKEYSCGLLNPLLLCNVQA

In some embodiments, one or more of the amino acids in the peptide aremodified to reduce proteolysis. In some embodiments, one or more of theamino acid residues in the peptide are methylated. In some embodiments,one or more of the asparagine amino acid residues in the peptide (if thepeptide comprises one or more asparagine amino acid residues) aremethylated. In some embodiments, one or more of the leucine amino acidresidues in the peptide (if the peptide comprises one or more leucineamino acid residues) are methylated. In some embodiments, the asparagineresidue at the amino acid position corresponding to position 11 of SEQID NO: 2 or 3 is methylated. In some embodiments, the leucine residue atthe amino acid position corresponding to position 13 of SEQ ID NO: 2 or3 is methylated.

Orthologs of proteins are typically characterized by possession ofgreater than 75% sequence identity counted over the full-lengthalignment with the amino acid sequence of specific protein using ALIGNset to default parameters. Proteins with even greater similarity to areference sequence will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 92%, at least 95%, or at least 98% sequence identity. Inaddition, sequence identity can be compared over the full length ofspecific domain (s) of the disclosed peptides. When significantly lessthan the entire sequence is being compared for sequence identity,homologous sequences will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85%, at least 90%, at least 95%, or atleast 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods are described at the NCSA Website.

Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue) as compared to any of SEQ ID NOs: 1-21. In someembodiments, the peptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 amino acid alterations as compared to any of SEQ ID NOs: 1-21.In some embodiments, the peptide having 1 or more amino acid alterationsas compared any of SEQ ID NOs: 1-21 is capable of still binding to an Fcreceptor (e.g., to an Fc receptor such as FcγRI).

In some embodiments, the peptide has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10conservative amino acid substitutions as compared to any of SEQ ID NOs:1-21. A “conservative amino acid substitution” is one in which an aminoacid residue is substituted by another amino acid residue having a sidechain (R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a peptide (e.g.,the peptide's ability to bind to an Fc receptor such as FcγRI). In caseswhere two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids thathave side chains with similar chemical properties include (1) aliphaticside chains: glycine, alanine, valine, leucine and isoleucine; (2)aliphatic-hydroxyl side chains: serine and threonine; (3)amide-containing side chains: asparagine and glutamine; (4) aromaticside chains: phenylalanine, tyrosine, and tryptophan; (5) basic sidechains: lysine, arginine, and histidine; (6) acidic side chains:aspartate and glutamate, and (7) sulfur-containing side chains arecysteine and methionine. In particular embodiments, conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445. A “moderately conservative” replacement is any changehaving a nonnegative value in the PAM250 log-likelihood matrix.

In some embodiments, one or more amino acid residues in any of thepeptide sequences disclosed herein are replaced with a non-naturallyoccurring or non-standard amino acid, modifying one or more amino acidresidue into a non-naturally occurring or non-standard form, orinserting one or more non-naturally occurring or non-standard amino acidinto the sequence. Examples of numbers and locations of alterations insequences of the invention are described elsewhere herein. Naturallyoccurring amino acids include the 20 “standard” L-amino acids identifiedas G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by theirstandard single-letter codes. In some embodiments, any of the peptidesdisclosed herein comprises one or more non-naturally occurring aminoacids. Non-standard amino acids include any other residue that may beincorporated into a peptide backbone or result from modification of anexisting amino acid residue. Non-standard amino acids may be naturallyoccurring or non-naturally occurring. Several naturally occurringnon-standard amino acids are known in the art, such as 4-hydroxyproline,5-hydroxylysine, 3-methylhistidine, N-acetylserine, etc. (Voet & Voet,Biochemistry, 2nd Edition, (Wiley) 1995). Those amino acid residues thatare derivatised at their N-alpha position will only be located at theN-terminus of an amino-acid sequence. Normally in the present inventionan amino acid is an L-amino acid, but it may be a D-amino acid.Alteration may therefore comprise modifying an L-amino acid into, orreplacing it with, a D-amino acid. Methylated, acetylated and/orphosphorylated forms of amino acids are also known, and amino acids inthe present invention may be subject to such modification.

Amino acid sequences in antibody domains and peptides of the inventionmay comprise non-natural or non-standard amino acids described above.Non-standard amino acids (e.g. D-amino acids) may be incorporated intoan amino acid sequence during synthesis, or by modification orreplacement of the “original” standard amino acids after synthesis ofthe amino acid sequence.

In some embodiments, use of non-standard and/or non-naturally occurringamino acids increases structural and functional diversity, and can thusincrease the potential for achieving desired properties in a peptide ofthe invention. Additionally, D-amino acids and analogues have been shownto have better pharmacokinetic profiles compared with standard L-aminoacids, owing to in vivo degradation of peptides having L-amino acidsafter administration to an animal e.g. a human.

In some embodiments, at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%of the amino acids in the peptide are D-amino acids. In particularembodiments, all of the amino acids in the peptide are D-amino acids. Insome embodiments, the disclosure provides for a peptide that comprisesan amino acid sequence that is in the reverse order of any of the aminoacid sequences disclosed herein or that is in the reverse order of anamino acid sequence portion of an antibody that is capable of binding toan Fc receptor (e.g., FcγRI). In some embodiments, the disclosureprovides for a “retro-inverse” peptide in which all of the amino acidsin the peptide are D-amino acids and that comprises an amino acidsequence that is in the reverse order of any of the amino acid sequencesdisclosed herein or that is in the reverse order of an amino acidsequence portion of an antibody that is capable of binding to an Fcreceptor (e.g., FcγRI). A “retro-inverse” peptide may be desirablebecause it would be less likely to be cleaved by proteases present in asubject that had been administered the peptide or a conjugate comprisingthe peptide. Peptides of the invention can be further modified orderivatized to contain additional nonproteinaceous moieties that areknown in the art and readily available. Such derivatives may improve thesolubility, absorption and/or biological half-life of the compounds. Themoieties may alternatively eliminate or attenuate any undesirableside-effect of the compounds.

Exemplary derivatives include compounds in which: the compound iscross-linked or is rendered capable of cross-linking between molecules.For example, the peptide portion may be modified to contain one Cysresidue and thereby be able to form an intermolecular disulfide bondwith a like molecule. The compound may also be cross-linked through itsC-terminus.

In some embodiments, the N-terminus may be acylated or modified to asubstituted amine. Exemplary N-terminal derivative groups include —NRR1(other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1,succinimide, or benzyloxycarbonyl-NH—(CBZ—NH—), wherein R and R1 areeach independently hydrogen or lower alkyl and wherein the phenyl ringmay be substituted with 1 to 3 substituents selected from the groupconsisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.

In some embodiments, the C-terminus may be esterified or amidated. Forexample, methods described in the art may be used to add(NH—CH₂—CH₂—NH₂)₂ to peptides of this invention. Likewise, methodsdescribed in the art may be used to add-NH2 to peptides of thisinvention. Exemplary C-terminal derivative groups include, for example,—C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 areindependently hydrogen or C1-C8 alkyl (e.g. C1-C4 alkyl).

In some embodiments, a disulfide bond may be replaced with another, morestable, cross-linking moiety (e.g., an alkylene). See, for example:Bhatnagar et al. (1996), J. Med. Chem. 39: 3814 9; Alberts et al. (1993)Thirteenth Am. Pep. Symp., 357-9.

In some embodiments, one or more individual amino acid residues may bemodified. Various derivatizing agents are known to react specificallywith selected side chains or terminal residues, as described in detailbelow:

In some embodiments, lysinyl residues and amino terminal residues may bereacted with succinic or other carboxylic acid anhydrides, which reversethe charge of the lysinyl residues. Other suitable reagents forderivatizing alpha-amino-containing residues include imidoesters such asmethyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4pentanedione; and transaminase-catalyzed reaction with glyoxylate.

In some embodiments, arginyl residues may be modified by reaction withany one or combination of several conventional reagents, includingphenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin.Derivatization of arginyl residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. N-acetylimidazole; and tetranitromethane may be usedto form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively.

In some embodiments, carboxyl sidechain groups (aspartyl or glutamyl)may be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4, 4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

In some embodiments, glutaminyl and asparaginyl residues may bedeamidated to the corresponding glutamyl and aspartyl residues.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisdisclosure. Cysteinyl residues can be replaced by amino acid residues orother moieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides of the invention or their functional derivatives to awater-insoluble support matrix or to other macromolecular vehicles.Commonly used cross-linking agents include, e.g., 1, 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimideesters, for example, esters with 4-azidosalicylic acid, homobifunctionalimidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such asbis-N-maleimido-1, 8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio] propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

In some embodiments, carbohydrate (oligosaccharide) groups mayconveniently be attached to sites that are known to be glycosylationsites in proteins. In some embodiments, O-linked oligosaccharides areattached to serine (Ser) or threonine (Thr) residues while N-linkedoligosaccharides are attached to asparagine (Asn) residues when they arepart of the sequence Asn-X-Ser/Thr, where X can be any amino acid exceptproline. In some embodiments, X is one of the 19 naturally occurringamino acids other than proline. The structures of N-linked and O-linkedoligosaccharides and the sugar residues found in each type aredifferent. One type of sugar that is commonly found on both isN-acetylneuraminic acid (referred to as sialic acid). Sialic acid isusually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycosylated compound.

Such site(s) may be incorporated in the linkers contemplated for thepeptides of the invention and, in some embodiments, are glycosylated bya cell during recombinant production of the peptide compounds (e.g., inmammalian cells such as CHO, BHK, COS). However, such sites may furtherbe glycosylated by synthetic or semi-synthetic procedures known in theart.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulphur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San I Francisco), pp. 79-86 (1983).

In particular embodiments, the moieties suitable for derivatization ofthe peptides are water soluble polymers. Non-limiting examples of watersoluble polymers include, but are not limited to, polyethylene glycol(PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone) polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The polymer may be linked to the peptide in the manner setforth in: U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417,4,791,192 or 4,179,337. WO 93/00109 also describes methods of linkingamino acid residues in peptides to PEG molecules. The number of polymersattached to the peptide may vary, and if more than one polymers areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the peptide to be improved or whether it willbe used in a therapy under defined conditions, for example.

In an alternative embodiment, peptides of the invention can be furthermodified to contain a serum carrier protein in order to extend the halflife in vivo. The serum carrier protein may be a naturally occurringserum carrier protein or a fragment thereof. Particular examples includethyroxine-binding protein, transthyretin, al-acid glycoprotein,transferrin, fibrinogen and especially, albumin, together with fragmentsthereof. In some embodiments, the carrier proteins are of human origin.Where desired each may have one or more additional or different aminoacids to the naturally occurring sequence providing that the resultingsequence is functionally equivalent with respect to half-life. Fragmentsinclude any smaller part of the parent protein that retains the carrierfunction of the mature sequence. The peptide and carrier proteincomponents may be directly or indirectly covalently linked. Indirectcovalent linkage is intended to mean that an amino acid in a peptide isattached to an amino acid in a carrier protein through an interveningchemical sequence, for example a bridging group. Particular bridginggroups include for example aliphatic, including peptide. Direct covalentlinkage is intended to mean that an amino acid in a peptide isimmediately attached to an amino acid in a carrier protein without anintervening bridging group. Particular examples include disulphide[—S—S—] and amide [—CONH—] linkages, for example when a cysteine residuein one component is linked to a cysteine residue in another through thethiol group in each, and when the C-terminal acid function of onecomponent is linked to the N-terminal amine of the other.

In addition to any of the targeting moieties described herein, any ofthe peptides disclosed herein may also be labelled with a detectable orfunctional label. Thus, a peptide can be present in the form of apeptide conjugate so as to obtain a detectable and/or quantifiablesignal. A peptide conjugate may comprise a peptide of the inventionconjugated with a detectable or functional label. A label can be anymolecule that produces or can be induced to produce a signal, includingbut not limited to fluorescers, radiolabels, enzymes, chemiluminescersor photosensitizers. Thus, binding may be detected and/or measured bydetecting fluorescence or luminescence, radioactivity, enzyme activityor light absorbance.

Suitable labels include, by way of illustration and not limitation,

-   -   enzymes, such as alkaline phosphatase, glucose-6-phosphate        dehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxydase,        glucose amylase, carbonic anhydrase, acetylcholinesterase,        lysozyme, malate dehydrogenase and peroxidase e.g. horseradish        peroxidase;    -   Fluorescent label/pigment/stain;        -   fluorescent labels or fluorescers, such as fluorescein and            its derivatives, fluorochrome, rhodamine compounds and            derivatives, GFP (GFP for “Green Fluorescent Protein”),            dansyl, umbelliferone, phycoerythrin, phycocyanin,            allophycocyanin, o-phthaldehyde, and fluorescamine;            fluorophores such as lanthanide cryptates and chelates e.g.            Europium etc (Perkin Elmer and Cis Biointernational),            -chemiluminescent labels or chemiluminescers, such as            isoluminol, luminol and the dioxetanes;    -   bio-luminescent labels, such as luciferase and luciferin;    -   sensitizers;    -   coenzymes;    -   enzyme substrates;    -   radiolabels including but not limited to bromineW, carbon14,        cobalt57, fluorineδ, gallium67, gallium 68, hydrogen3 (tritium),        indium111, indium 113m, iodine123m, iodine125, iodine126,        iodine131, iodine133, mercury107, mercury203, phosphorous32,        rhenium99m, rhenium101, rhenium105, ruthenium95, ruthenium97,        ruthenium103, ruthenium105, scandium47, selenium75, sulphur35,        technetium99, technetium99m, tellurium121m, tellurium122m,        tellurium125m, thulium165, thulium167, thulium168, yttrium199        and other radiolabels mentioned herein;    -   particles, such as latex or carbon particles; metal sol;        crystallite; liposomes; cells, etc., which may be further        labelled with a dye, catalyst or other detectable group;        -molecules such as biotin, digoxygenin or 5-bromodeoxyuridine;    -   toxin moieties, such as for example a toxin moiety selected from        a group of Pseudomonas exotoxin (PE or a cytotoxic fragment or        mutant thereof), Diphtheria toxin or a cytotoxic fragment or        mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a        cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic        fragment thereof, saporin or a cytotoxic fragment thereof,        pokeweed antiviral toxin or a cytotoxic fragment thereof and        bryodin 1 or a cytotoxic fragment thereof.

Suitable enzymes and coenzymes are disclosed in Litman, et al., U.S.Pat. No. 4,275,149, and Boguslaski, et al., U.S. Pat. No. 4,318,980,each of which are herein incorporated by reference in their entireties.Suitable fluorescers and chemiluminescers are disclosed in Litman, etal., U.S. Pat. No. 4,275,149, which is incorporated herein by referencein its entirety. Labels further include chemical moieties, such asbiotin that may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin or streptavidin. Detectable labels may beattached to peptides of the invention using conventional chemistry knownin the art, or by gene fusion.

In some embodiments, the disclosure provides for a peptide that is anantibody fragment capable of binding to an Fc receptor (e.g., an FcγRI).It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward et al 1989, McCafferty et al 1990, Holt et al 2003),which consists of a VH or a VL domain; (v) isolated CDR regions; (vi)F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al 1988,Huston et al 1988); (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; Hollinger et al 1993).Fv, scFv or diabody molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al 1996).Minibodies comprising a scFv joined to a CH3 domain may also be made (Huet al 1996). Other examples of binding fragments are Fab′, which differsfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain, including one or more cysteinesfrom the antibody hinge region, and Fab′-SH, which is a Fab′ fragment inwhich the cysteine residue (s) of the constant domains bear a free thiolgroup. Antibody fragments that comprise an antibody antigen-binding siteinclude, but are not limited to, molecules such as Fab, Fab′, Fab′-SH,scFv, Fv, dAb and Fd. Various other antibody molecules including one ormore antibody antigen-binding sites have been engineered, including forexample Fab2, Fab3, diabodies, triabodies, tetrabodies and minibodies.Antibody molecules and methods for their construction and use aredescribed in Hollinger & Hudson (2005).

2. The Targeting Moiety

In some embodiments, any of the peptides disclosed herein is conjugatedto one or more targeting moieties.

In some embodiments, the targeting moiety targets protein aggregates. Insome embodiments, the protein aggregates comprise aggregates of any oneof the following proteins: alpha-synuclein, tau, amyloid beta, TDP-43,SOD1, FUS, TDP-43, prion protein, immunoglobulin light chain,transthyretin, fibrinogen, collagen, islet amyloid polypeptide,lysozyme, calcitonin, serum amyloid A protein, LECT2, amylin, gelsolin,fibrinogen A, prolactin, keratoepithelin, and β₂-microglobin.

In some embodiments, the targeting moiety targets lipid aggregates. Insome embodiments, the lipid aggregates are cholesterol aggregates.

In some embodiments, the targeting moiety targets polysaccharideaggregates. In some embodiments, the targeting moiety targets glycogenaggregates.

In some embodiments, the targeting moiety is a peptide. In someembodiments, the targeting moiety is a nucleic acid. In someembodiments, the targeting moiety is a lipid. In some embodiments, thetargeting moiety is a polysaccharide. In particular embodiments, thetargeting moiety is a dye, or a derivative thereof.

In some embodiments, the targeting moiety is a small molecule. In someembodiments, the targeting moiety is less than 1000 g/mol, less than 900g/mol, less than 800 g/mol, less than 700 g/mol, less than 800 g/mol,less than 700 g/mol, less than 600 g/mol, less than 500 g/mol, less than400 g/mol, less than 300 g/mol, less than 200 g/mol, or less than 100g/mol in size. In particular embodiments, the targeting moiety is lessthan 500 g/mol in size. In particular embodiments, the targeting moietyis less than 400 g/mol in size.

In particular embodiments, the targeting moiety is a dye, or aderivative thereof. As used herein, a “dye” is defined as being acompound that is, or is derived from, a natural or synthetic substanceused to add a color to or change the color of protein, polysaccharides,lipid, metals and/or nucleotides. In some embodiments, the dye may beused to stain cells or components thereof. The skilled worker wouldunderstand what classifies as a dye/stain. In some embodiments, the dyeis a compound capable of binding to a protein, polysaccharide, lipid,metal, and/or nucleotide to facilitate visualization of the protein,polysaccharide, lipid, metal, and/or nucleotide. In preferredembodiments, the dye binds to an aggregate or a component present in anaggregate. In some embodiments, the dye binds to protein,polysaccharide, lipid, metal, and/or nucleotide in an aggregate. In someembodiments, the targeting moiety is a derivative of a dye that retainsthe ability to bind to a specific target (e.g., proteins, peptides,polysaccharides, nucleotides, lipids or aggregates thereof), but lacksor has a modified staining capability as compared to the dye from whichthe derivative was derived.

In some embodiments, the dye binds to amyloid. In some embodiments, thedye is selected from the group consisting of: a thioflavin, pentamerformyl thiophene acetic acid (pFTAA), heptamer formyl thiophene aceticacid (hFTAA), Congo Red, crystal violet, 1-amino-8-naphtalene sulphonate(ANS), 4-(dicyanovinyl)-julolidine (DCVJ), aminobenzanthrone, MethoxyXO4, thiazin red, Pittsburgh B, ProteoStat® and amyloid-bindingderivatives thereof. In some embodiments, the thioflavin is thioflavinT, or an amyloid-binding derivative thereof. In some embodiments, theamyloid-binding derivative of thioflavin T comprises the formula ofCompound I:

In some embodiments, the amyloid comprises any one or more of thefollowing proteins: alpha-synuclein, tau, amyloid beta, TDP-43, SOD1,FUS, C79ORF, TDP-43, prion protein, immunoglobulin light chain,transthyretin, fibrinogen, collagen, islet amyloid polypeptide,lysozyme, calcitonin, serum amyloid A protein, LECT2, amylin, gelsolin,fibrinogen A, prolactin, keratoepithelin, and β₂-microglobin, orfragments or derivatives thereof. In some embodiments, the amyloidcomprises lysozyme.

In some embodiments, the dye binds to cholesterol. In some embodiments,the dye is filipin, or a cholesterol-binding derivative thereof.

In some embodiments, the dye binds to collagen. In some embodiments, thedye is sinus red, Col-F, Oregon Green 488, Picrosirius red. Light GreenSF yellowish, Fast Green FCF, methyl blue, water blue, aniline blue, andcollagen-binding derivatives thereof.

In some embodiments, the dye binds polysaccharide aggregates. In someembodiments, the dye binds glycogen aggregates. In some embodiments, thedye is selected from the group consisting of carmine, periodic acid,alcian blue, and Dimethyl methylene blue.

In some embodiments, the dye binds nucleotide aggregates. In someembodiments, the dye binds to RNA aggregates. In some embodiments, thedye binds DNA aggregates. In some embodiments, the dye binds topolynucleotides having expanded CUG, CAG, CCUG, CCG or CGG repeats.

In some embodiments, the targeting moiety has been modified tofacilitate conjugation to the peptide. In some embodiments, thetargeting moiety has been modified such that it includes a carboxylgroup. In some embodiments, the carboxyl group of the targeting moietyis bonded to the amino terminus of the peptide. In some embodiments, thetargeting moiety has been modified such that it includes an amino group.In some embodiments, the amino group of the targeting moiety is bondedto the carboxy terminus of the peptide. In some embodiments, thetargeting moiety is conjugated to the peptide by means of a linker. Insome embodiments, the linker is selected from the group consisting ofnon-cleavable linker, hydrazone linkers, thioether linkers, disulfidelinkers, peptide linkers and β-glucuronide linkers. In certain suchembodiments, the linker is rigid.

3. The Conjugates

In some embodiments, any of the peptides disclosed herein is fused toany of the targeting moieties disclosed herein to generate any of theconjugates disclosed herein.

In some embodiments, any of the targeting moieties disclosed herein(e.g., any of the dyes disclosed herein) are conjugated directly to anyof the peptides disclosed herein (e.g., in the absence of a linker). Insome embodiments, any of the targeting moieties disclosed herein (e.g.,any of the dyes disclosed herein) are conjugated to any of the peptidesdisclosed herein by means of a linker or linkage moiety. In someembodiments, any of the targeting moieties disclosed herein (e.g., anyof the dyes disclosed herein) are covalently attached by linkages to thepeptide. In some embodiments, the linker is rigid. In alternativeembodiments, the linker is flexible. In preferred embodiments, thelinker should not (i) inhibit and/or adversely affect theaggregate-targeting properties of the targeting moiety (e.g., dye), or(ii) inhibit and/or adversely affect the ability of the peptide tocompete with an Fc fragment of an IgG for binding to an Fc receptor.

In some embodiments, the targeting moiety includes a reactive linkinggroup, “linking moiety”, at one of the substituent positions forcovalent attachment of the targeting moiety (e.g., dye) to a peptide.Linking moieties capable of forming a covalent bond are typicallyelectrophilic functional groups capable of reacting with nucleophilicmolecules, such as alcohols, alkoxides, amines, hydroxylamines, andthiols. Examples of electrophilic linking moieties include succinimidylester, isothiocyanate, sulfonyl chloride, sulfonate ester, silyl halide,2, 6-dichlorotriazinyl, pentafluorophenyl ester, phosphoramidite,maleimide, iodoacetamide, haloacetyl, epoxide, alkylhalide, allylhalide, aldehyde, ketone, acylazide, and anhydride. In some embodiments,a linking moiety is an N-hydroxysuccinimidyl ester (NHS) of a carboxylgroup substituent on a targeting moiety (e.g., dye).

In some embodiments, the linking moiety is a phosphoramidite reagent anyof the dyes disclosed herein. Other activating and coupling reagentsinclude TBTU (2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH(N,N′,N″,N′″-tetramethyluronium2-fluoro-hexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and arylsulfonylhalides, e.g. triisopropylbenzenesulfonyl chloride.

In some embodiments, the linker comprises an atom such as oxygen orsulphur, a unit such as —NH—, —CH₂—, —C(O)—, —C(O)NH—, or a hydrocarbylgroup. The term “hydrocarbyl group” refers to an optionally substituted,linear or cyclic, straight or branched carbon chain that may be fullysaturated or have one or more units of unsaturation. In certainembodiments, the hydrocarbyl group comprises one or more units ofunsaturation. In some embodiments, the linker comprises a saturated orunsaturated C₁₋₃₀ hydrocarbyl group which is optionally substituted. Insome embodiments, the linker comprises a saturated or unsaturated C₁₋₂₅hydrocarbyl group which is optionally substituted. In some embodiments,the linker comprises a saturated or unsaturated C₁₋₂₀ hydrocarbyl groupwhich is optionally substituted. In some embodiments, the linkercomprises a saturated or unsaturated C₁₋₁₅ hydrocarbyl group which isoptionally substituted. In some embodiments, the linker comprises asaturated or unsaturated C₁₋₁₀ hydrocarbyl group which is optionallysubstituted. In some embodiments, the linker comprises a saturated orunsaturated C₁₋₈ hydrocarbyl group which is optionally substituted. Insome embodiments, the linker comprises a saturated or unsaturated C₁₋₆hydrocarbyl group which is optionally substituted. In some embodiments,the linker comprises a saturated or unsaturated C₁₋₄ hydrocarbyl groupwhich is optionally substituted. In some embodiments, the linkercomprises a saturated or unsaturated C₁₋₂ hydrocarbyl group which isoptionally substituted. In some embodiments of any of the foregoing, oneor more saturated carbons of the chain are optionally replaced by—C(O)—, —C(O)C(O)—, —CONH—, —CONHNH—, —CO₂—, —OC(O)—, —NHCO₂—, —O—,—NHCONH—, —OC(O)NH—, —NHNH—, —NHCO—, —S—, —SO—, —SO₂—, —NH—, —SO₂NH—, or—NHSO₂—. In certain embodiments of the foregoing, the linker is rigid.

In some embodiments, any of the targeting moieties disclosed herein(e.g., any of the dyes disclosed herein) is covalently linked to any ofthe peptides disclosed herein by means of a linker comprising thestructure of Compound II.

wherein n is 1-30, and wherein m is 0, 1, 2, 3, 4, or 5. In someembodiments, n is 1-25, 1-20, 1-15, 1-10, 1-8, 1-6, 1-4, 1-2 or 1. Insome embodiments, n is 2. In some embodiments, m is 0.

In some embodiments, the linker is a β-Ala linker, a short polyethyleneglycol (PEG) linker, or a long PEG linker. In particular embodiments,the linker is a β-alanine linker. In some embodiments, the PEG linkercomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 units of PEG. In particular embodiments,the linker is β-alanine; 9-amino-4,7-dioxanonanoic acid or6-amino-4-oxaohexanoic acid. In some embodiments, the linker is an FMOCderivatized linker.

In some embodiments, the conjugate comprises the structure of CompoundIII:

wherein R1 is biotin or hydrogen;wherein R² comprises:

In some embodiments, R¹ is biotin. In some embodiments, R¹ is hydrogen.In particular embodiments, R² comprises

Peptides can be conjugated to a targeting moiety (e.g., dye) at sitesincluding an amino acid side-chain, the amino terminus, and the carboxyterminus. Peptides can be functionalized to bear reactive amino, thiol,sulfide, disulfide, hydroxyl, and carboxyl groups at any of these sites.

Any of the conjugates disclosed herein can be prepared by methods knownto the person skilled in the art. Peptides comprised of amino acids andamino acid analogs may be covalently fused to a targeting moiety byconjugation with any of the dyes disclosed herein. In some embodiments,the dye bears an electrophilic linking moiety which reacts with anucleophilic group on the peptide, e.g. amino terminus, or side-chainnucleophile of an amino acid. Alternatively, the dye may be innucleophilic form, e.g. amino- or thiol-linking moiety, which reactswith an electrophilic group on the peptide, e.g. NHS of the carboxylterminus or carboxyl side-chain of an amino acid. In some embodiments,the dye may be on a solid support, i.e. synthesis resin, during theconjugate reaction. Alternatively, the peptide may have been cleavedprior to conjugate to the dye. In certain embodiments, certain aminoacid side-chains may allow conjugation with activated forms of any ofthe dyes disclosed herein. Aspartic acid, glutamic acid, lysine,arginine, cysteine, histidine, and tyrosine have reactive functionalityfor conjugations. By appropriate selection of protecting groups, certainreactive functionality on the peptide can be selectively unmasked forreaction with a targeting moiety (e.g., dye).

In some embodiments, peptides can be coupled to enzymes or tofluorescent labels directly or by the intermediary of a spacer group orof a linking group, such as a polyaldehyde, like glutaraldehyde,ethylenediaminetetraacetic acid (EDTA), diethylene-triaminepentaaceticacid (DPTA), or in the presence of coupling agents. Conjugatescontaining labels of fluorescein type can be prepared by reaction withan isothiocyanate.

In some embodiments, the methods known to the person skilled in the artexisting for coupling the radioisotopes to the peptides either directlyor via a chelating agent, such as EDTA, DTPA mentioned above can be usedfor the radioelements which can be used in diagnosis. It is likewisepossible to perform labelling with sodium¹²⁵ by the chloramine T method[i] or else with technetium^(99m) by the technique of Crockford et al.,or attached via DTPA as described by Hnatowich.

In some embodiments, any of the conjugates of the disclosure bind to,for example, an Fc receptor (e.g., FcγRI). Such binding may take placein vivo, e.g. following administration of a conjugate, or it may takeplace in vitro, for example in ELISA, Western blotting,immunocytochemistry, immunoprecipitation, affinity chromatography, andbiochemical or cell-based assays. Those skilled in the art are able tochoose a suitable mode of determining binding of the peptide to the Fcreceptor (e.g., FcγRI) according to their preference and generalknowledge, in light of the methods disclosed herein. Such methods mayinclude inter alia competitive ELISA and alpha screen.

In some embodiments, the disclosure provides for a dimer or oligomer ofany of the conjugates disclosed herein. In some embodiments, theconjugates in the dimer or oligomer are linked to each other by means ofa linker. In some embodiments, the peptide in the conjugate binds to twohFcγRIs. In some embodiments, the peptide in the conjugate activates Fceffector function. In some embodiments, the conjugate comprises two Fcmimetic peptides linked by a KKKKK linker (SEQ ID NO: 22).

In some embodiments, any of the conjugates disclosed herein is capableof recruiting an immune cell to the molecular aggregates. In someembodiments, the immune cell is a monocyte-derived cell. In someembodiments, the immune cell is a macrophage. In some embodiments, theimmune cell is a monocyte. In some embodiments, the immune cell is aglial cell (e.g., a microglial cell). In some embodiments, the conjugatebinds to the immune cell and facilitates activation of the immune cell.In some embodiments, the conjugate facilitates phagocytosis of at leasta portion of the molecular aggregates.

4. Methods of Treatment

For any of the methods described herein, the disclosure contemplates theuse of any of the conjugates and/or compositions described throughoutthe application. In addition, for any of the methods described herein,the disclosure contemplates the combination of any step or steps of onemethod with any step or steps from another method.

The terms “treatment”, “treating”, and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. “Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease, andincludes administration of a composition which reduces the frequency of,or delays the onset of, symptoms of a medical condition in a subject inneed relative to a subject which does not receive the composition.“Treatment” as used herein covers any treatment of a disease orcondition of a mammal, particularly a human, and includes: (a)preventing symptoms of the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet begun experiencing symptoms; (b) inhibiting the disease or condition(e.g., arresting its development); or (c) relieving the disease orcondition (e.g., causing regression of the disease or condition,providing improvement in one or more symptoms). For example, treating anamyloidosis disease such as Alzheimer's Disease encompasses preventionof significant memory loss or cognitive decline, reduction in amyloidplaques, improvement in memory, and/or improvement or reduction ofcognitive decline. Treatment of other amyloidosis conditions mightencompass prevention/alleviation/reversal of diarrhea, weight loss,fatigue, glossomegaly, haemorrhage, somatic sensory deficits, posturalhypotension, peripheral edema and splenomegaly. Treatment ofatherosclerosis or heart disease might encompass clearance ofcholesterol/lipid aggregates and/or prevention/alleviation/reversal ofatherosclerosis and/or heart disease. Treatment of glycogen storagediseases might encompass clearance of glycogen deposits and/or anyassociated symptoms associated with that disease (locomotor defects,cognitive defects, hepatic defects, etc.).

In some embodiments, the disclosure provides for a method of recruitingan immune cell to an aggregate by contacting the aggregate with any ofthe conjugates disclosed herein. In some embodiments, the immune cell isany of the immune cells disclosed herein. In some embodiments, theimmune cell is activated such that it is capable of clearing theaggregate (e.g., by phagocytosis or by recruiting/activating additionalimmune cells). In some embodiments, the aggregate is any of theaggregates disclosed herein. In some embodiments, the aggregate andimmune cell are in vitro. In some embodiments, the aggregate and immunecell are in vivo. In some embodiments, the aggregate and immune cell arein an animal. In some embodiments, the animal is a mammal. In someembodiments, the mammal is a human. In some embodiments, the immune cellis a monocyte-derived cell. In some embodiments, the immune cell is amacrophage. In some embodiments, the immune cell is a monocyte. In someembodiments, the immune cell is a microglial cell.

In some embodiments, the disclosure provides for a method of treating asubject in need thereof with any one or more of the conjugates disclosedherein, wherein the subject has a disease or disorder associated withprotein aggregates. In some embodiments, the protein aggregates compriseany one or more of alpha-synuclein, tau, amyloid beta, TDP-43, SOD1,FUS, TDP-43, prion protein, immunoglobulin light chain, transthyretin,fibrinogen, collagen, islet amyloid polypeptide, lysozyme, calcitonin,serum amyloid A protein, LECT2, amylin, gelsolin, fibrinogen A,prolactin, keratoepithelin, and β₂-microglobin, or aggregate-formingfragments or derivatives thereof. In some embodiments, the amyloid iscomposed of repetitive peptide sequences resulting from RAN-translationof nucleotide repeat expansions as seen in C9ORF72, Huntington'sDisease, Fragile XA, Fragile XE, Friedrich's ataxia, Myotonic Dystrophytype 1 (DM1), Creutzfeldt-Jakob Disease, Myotonic Dystrophy type 2(DM2), and Spinocerebellar ataxia types 8, 10, and 12 (SCAB, SCA10, SCA12).

In some embodiments, the disease or disorder is an amyloidosis. In someembodiments, the amyloidosis is selected from the group consisting of:AL amyloidosis, AA amyloidosis, Alzheimer Disease, LECT2 amyloidosis,leptomeningeal amyloidosis, senile systemic amyloidosis, familialamyloid polyneuropathy, haemodialysis-associated amyloidosis, type 2diabetes, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinkersyndrome, Finnish type amyloidosis, cerebral amyloid angiopathy,familial visceral amyloidosis, primary cutaneous amyloidosisprolactinoma, familial corneal amyloidosis, Parkinson's Disease,amyotrophic lateral sclerosis, cerebral amyloid angiopathy,frontotemporal lobar dementia, medullary thyroid carcinoma, and B2Mamyloidosis. In some embodiments, the amyloidosis is a familialamyloidosis. In some embodiments, the amyloidosis is systemicamyloidosis.

In some embodiments, the disclosure provides for a method of treating asubject in need thereof with any one or more of the conjugates disclosedherein, wherein the subject has a disease or disorder associated withpolysaccharide aggregates. In some embodiments, the polysaccharide isglycogen. In some embodiments, the disease or disorder is a glycogenstorage disease or disorder. In some embodiments, the disease ordisorder is selected from the group consisting of Pompe Disease, vonGierke Disease, Forbes-Cori Disease, Andersen Disease, McArdle Disease,Hers Disease, Tarui's Disease, Fanconi-Bickel Syndrome, GSD IX, GSD X,GSD XI, GSD XII, GSD XIII, GSD XV, and Lafora Disease.

In some embodiments, the disclosure provides for a method of treating asubject in need thereof with any one or more of the conjugates disclosedherein, wherein the subject has a disease or disorder associated withlipid aggregates. In some embodiments, the lipid is cholesterol. In someembodiments, the lipid is sphingomyelin. In some embodiments, the lipidis globotriaosylceramide (Gb3) or glucocerebroside. In some embodiments,the disease or disorder is heart disease or atherosclerosis. In someembodiments, the disease or disorder is hypercholesterolemia. In someembodiments, the disease or disorder is Alzheimer's Disease orParkinson's disease. In some embodiments, the disease or disorder isFabry disease or Niemann-Pick Type A, Type B or Type C disease.

In some embodiments, the disclosure provides for a method of treating asubject in need thereof with any one or more of the conjugates disclosedherein, wherein the subject has a disease or disorder associated withnucleotide aggregates. In some embodiments, the disease or disorder isassociated with RNA aggregates. In some embodiments, the disease ordisorder is associated with DNA aggregates. In some embodiments, thedisease or disorder is associated with aberrant microsateliteexpansions. Conditions associated with aberrant microsatellite expansion(also referred to herein as microsatellite expansion diseases) include anumber of neurological and neuromuscular diseases (O'Donnell, et al.,2002, Annu. Rev. Neurosci. 25: 315). These diseases or conditions arecaused by microsatellite repeat expansions in coding and non-codingregions. These microsatellite repeat expansions may include, forexample, CAG or CUG repeats, or variations thereof (e.g. CCUG). Forexample, the characterized coding region expansion diseases includeDentatorubral pallidoluysian atrophy (DRPLA), Huntington chorea (HD),Oculopharyngeal muscular dystrophy (OPMD), Spinobulbar muscular atrophy(SBMA), specific forms of Creutzfeldt-Jakob Disease and Spinocerebellarataxia types 1, 2, 3, 6, 7, and 17 (SCA1, SCA2, SCA3, SCA6, SCAT, SCA17). The characterized non-coding region expansion diseases includeFragile XA, Fragile XE, Friedrich's ataxia, Myotonic Dystrophy type 1(DM1), Myotonic Dystrophy type 2 (DM2), and Spinocerebellar ataxia types8, 10, and 12 (SCAB, SCA10, SCA 12). Huntington's disease andHuntington's disease-like type 2 (HDL2) are also caused by amicrosatellite expansion. Amyotrophic lateral sclerosis or fronotemporaldementia (FTD) are associated with repeat expansions in the chromosome 9open reading frame 72 (C9ORF72) gene.

In some embodiments, any of the conjugates described herein may becombined with an alternative therapy. It will be appreciated thattreatment of protein aggregation associated diseases according to thepresent invention may be combined with other treatment methods known inthe art (i.e., combination therapy). Thus, in the cases for amyloidosis,compounds of the present invention may be co-administered(simultaneously or separately) with additional drugs such asanti-amyloid drugs. Examples of such anti-amyloid drugs include, but arenot limited to, amyloid destabilizing antibodies, amyloid destabilizingpeptides and anti-amyloid small molecules. Other combination therapiesmay include a therapeutically effective amount of at least one drugselected from the group consisting of BACE inhibitors; muscarinicantagonists; cholinesterase inhibitors; gamma secretase inhibitors;gamma secretase modulators; HMG-CoA reductase inhibitors; non-steroidalanti-inflammatory agents; N-methyl-D-aspartate receptor antagonists;anti-amyloid antibodies; vitamin E; nicotinic acetylcholine receptoragonists; CB1 receptor inverse agonists or CB1 receptor antagonists; anantibiotic; growth hormone secretagogues; histamine H3 antagonists; AMPAagonists; PDE4 inhibitors; GABAA inverse agonists; inhibitors of amyloidaggregation; glycogen synthase kinase beta inhibitors; promoters ofalpha secretase activity; PDE-10 inhibitors; Exelon (rivastigmine);Cognex (tacrine); Tau kinase; anti-Abeta vaccine; APP ligands; agentsthat upregulate insulin cholesterol lowering agents; cholesterolabsorption inhibitors; fibrates; LXR agonists; LRP mimics; nicotinicreceptor agonists; H3 receptor antagonists; histone deacetylaseinhibitors; hsp90 inhibitors; ml muscarinic receptor agonists; 5-HT6receptor antagonists; mGluR1; mGluR5; positive allosteric modulators oragonists; mGluR2/3 antagonists; anti-inflammatory agents that can reduceneuroinflammation; Prostaglandin EP2 receptor antagonists; PAI-1inhibitors; and agents that can induce Abeta efflux.

Other combination therapies may include a therapeutically effectiveamount of at least one therapy selected from the group consisting ofphysical therapy, massage, cannabinoids (See, e.g., Ramirez, et al, TheJournal of Neuroscience, Feb. 23, 2005, 25(8):1904-1913); dimebon (See,e.g., R S Doody, et al., The Lancet 372:207-215 (2008); Selectiveestrogen receptor molecules (SERMs), e.g., raloxifene (EVISTA®);antihypertensives, including alpha-blockers, beta-blockers, alpha-betablockers, angiotensin-converting enzyme inhibitors, angiotensin receptorblockers (ARBs, such as valsartan (e.g., DIOVAN®)), calcium channelblockers, and diuretics (See, e.g., I Hajjar, et al, The Journals ofGerontology Series A: Biological Sciences and Medical Sciences 60:67-73(2005)); and antioxidants such as garlic extract, curcumin, melatonin,resveratrol, Ginkgo biloba extract, green tea, vitamin C and vitamin E(See, e.g., B Frank, et al., Ann Clin Psychiatry 17(4):269-86 (2005)).

In cases of cholesterol related diseases or disorders, any of theconjugates described herein may be combined with cholesterol-loweringand/or heart protective drugs such as statins, e.g., atorvastatin(LIPITOR®), cerivastatin (BAYCOL®), fluvastatin (e.g., LESCOL®),mevastatin, pitavastatin (e.g., LIVALO®), pravastatin (e.g.,PRAVACHOL®), rosuvastatin (e.g., CRESTOR®) and simvastatin (e.g.,ZOCOR®).

5. Pharmaceutical Compositions

In some embodiments, any of the conjugates described herein may beformulated into pharmaceutical compositions. Pharmaceutical compositionsfor use in accordance with the present disclosure may be formulated inconventional manner using one or more physiologically acceptablecarriers or excipients. Such formulations will generally besubstantially pyrogen-free, in compliance with most regulatoryrequirements.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the conjugates which mayalso optionally be included in the composition as described above, maybe administered simultaneously or sequentially with the subjectcompounds in the methods disclosed herein.

In some embodiments, conjugates disclosed herein will be administeredparentally, and particularly intravenously or subcutaneously. In someembodiments, the conjugates are administered intrathecally. In someembodiments, the conjugates are administered intramuscularly. In someembodiments, the conjugates are administered intracerebroventrically. Insome embodiments, the conjugates are administered intranasally.Pharmaceutical compositions suitable for parenteral administration maycomprise one or more conjugates in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site. In certain embodiments, compositionsof the present invention may include a matrix capable of delivering oneor more conjugates to a target tissue site, providing a structure forthe developing tissue and optimally capable of being resorbed into thebody. For example, the matrix may provide slow release of theconjugates. Such matrices may be formed of materials presently in usefor other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalcium phosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administeredorally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject conjugates of the invention. The various factors include,but are not limited to, the patient's age, sex, and diet, the severitydisease, time of administration, and other clinical factors. Optionally,the dosage may vary with the type of matrix used in the reconstitutionand the types of compounds in the composition. The addition of otherknown growth factors to the final composition, may also affect thedosage. Progress can be monitored by periodic assessment of bone growthand/or repair, for example, X-rays (including DEXA), histomorphometricdeterminations, and tetracycline labeling.

Another targeted delivery system for conjugates is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. In some embodiments, the colloidal system of this inventionis a liposome. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments of thepresent invention, and are not intended to limit the invention.

Example 1: Generation of BDB

Compound 1 is a carboxylic acid derivative of thioflavin T. This isadded as the terminal residue to the biotinylated synthetic cyclicpeptide shown in FIG. 1, which is a derivative of the cp33 peptide(Bonetto S et al. FASEB J. 2009; 23(2):575-85). The resulting conjugateis referred to as “BDB” in the Examples provided below.

Example 2: Determining the Binding of BDB to Amyloids

The binding of BDB to various amyloid proteins was evaluated in vitrousing a fluorometric assay. Results are summarized in FIG. 2.

The amyloid proteins (hen lysozyme (hLys), human full-length alphasynuclein, and human tau P301S) were individually prepared for use inthe fluorometric assay. hLys (Sigma) was dissolved in 136.8 mM NaCl,2.68 mM KCl, 0.01% w/v NaN₃, pH 2 at a concentration of 1.45 mg/ml andincubated at 55° C. for 24 h in an orbital shaker at 150 rpm. Human fulllength alpha synuclein (Alexotech) was dissolved in 150 mM NaCl, 1% v/vtriton-x100 in PBS at a concentration of 2.2 mg/ml and incubated at 37°C. for 72 h in an orbital shaker at 250 rpm. Human tau P301S wasincubated at 8 mg/ml with 4 mg/ml heparin (Sigma) in PBS, 30 mM3-(N-morpholino) propanesulfonic acid (MOPS) (pH 7.2) at 37° C. for 72h. The solution was mixed with nine volumes of PBS with 1% v/v sarkosyl(Sigma) and left rocking for 1 h at room temperature. Insoluble tau waspelleted by ultracentrifugation for 1 h at 4° C. and the pelletresuspended in PBS and sonicated at 100 W for 3×20 s (Hielscher UP200Stultrasonicator).

The binding of the BDB to amyloid was detected using a fluorometricassay. The amyloid binding was detected with the use of one of threefluorescent reporters solutions (30 μM BDB, cp33 peptide (Bonetto S etal. FASEB J. 2009; 23(2):575-85) or Thioflavin T (ThT) (Sigma) solution,in PBS). One microlitre of amyloid suspension (30 μM tau or lysozyme or150 μM α-synuclein) was mixed with 19 μl of one of three reportersolutions in black 384 well polystyrene plates (Greiner Bio-one). AnEnvision plate reader was set to excite at 450 nm and measure emissionwithin the range 460-680 nm. The corresponding fluorescence signals ofthe reporter solutions were subtracted from the experimental data. Asdemonstrated in FIG. 2, BDB and Tht have increased fluorescence in thepresence of amyloid proteins. The cp33 peptide, not conjugated withCompound 1, did not show a fluorescence signal in the presence ofamyloid.

Example 3: Determining the Binding of BDB to Activated Phagocytic Cells

In order to determine the binding affinity of BDB to activatedphagocytic cells, activated and non-activated U937 cells or murineprimary macrophages were resuspended in the presence of BDB,biotinylated human IgG1, or PBS. The cells were then stained usingeither AF488-streptavidin conjugate, APC-Cy7 dead/live stain, ordead/live stain only and measured using fluorescence activated cellsorting (FACS).

Specifically, U937 cells were cultured in RPMI 1640+2 mM Glutamine+10%v/v foetal bovine serum (FCS) for 48 h in the absence or presence of 100μg/ml human IFNγ (Thermo Fisher Scientific). Murine primary macrophageswere cultured for 48 h in the absence or presence of 100 μg/ml LPS(Sigma). Aliquots of 5×10⁵ cells were centrifuged 3 min at 300 g andresuspended either in 50 μl of 10 μM BDB, 1 μM biotinylated human IgG1or PBS. Samples were incubated at 4° C. for 1 h and then centrifuged for3 min at 100 g. Cells were resuspended in 50 μl of eitherAF488-streptavidin conjugate (Thermo Fisher Scientific, 1:500 in PBS)with APC-Cy7 dead/live stain (Thermo Fisher Scientific, 1:2000 in PBS)or dead/live stain only. Samples were incubated at 4° C. for 30 min andthen centrifuged for 3 min at 100 g. Samples were washed with 200 μlcold PBS and resuspended in 250 μl PBS for analysis using a FACS CantoII, with 10⁴ events recorded per condition. Using FlowJo software, cellswere visualised using forward and side scatter plots (FSC vs. SSC) andlive cells were gated according to their live/dead staining. Thepercentage of live cells positive for AF488 signal was identified foreach condition and plotted. The experiment was repeated three times andresults present as the mean (+/−SD). A one-way ANOVA was used estimatethe significance of differences between the conditions (** P<0.01, ****P<0.0001). As demonstrated in FIG. 3, U937 cells treated with IFNγ(IFNg) or murine primary macrophages treated with LPS, showed enhancedbinding of BDB. These results are consistent with BDB binding to theincreased surface expression of Fc receptors. IgG1 binds multiplereceptors with high affinity and labels cells irrespective of activationstatus.

Example 4: Titrating BDB and IgG1 Binding to IFNg Stimulated U937 Cells

The binding of BDB and IgG1 to IFNγ stimulated U937 cells was determinedusing the following methods. U937 cells were cultured in RPMI 1640+2 mMGlutamine+10% v/v FCS for 48 h in the presence of 100 μg/ml human IFNγ.Aliquots of 5×10⁵ U937 cells were centrifuged 3 min at 300 g andresuspended in 50 μl of either BDB or biotinylated IgG1 atconcentrations ranging from 0.01-5 μM or 0.01-1 μM respectively. Thestaining and FACS analysis were performed as described above.

FIG. 4 demonstrates that as the concentration of BDB increased, as manyas 80% of IFNg-activated U937 cells were bound. Half-maximal binding wassee at approximately 200 nM BDB. IgG1 bound to all cells under the sameconditions.

Example 5: Competing the Binding of BDB and Cp33 Peptide to Fc-ReceptorsUsing Unlabelled Human IgG1

A competitive assay between human IgG1 and BDB or cp33 peptide wasconducted in order to determine the specific binding of BDB and cp33peptides to the Fc receptor. Specifically, U937 cells were cultured inRPMI 1640+2 mM Glutamine+10% v/v FCS for 48h in the presence of 100μg/ml human IFNγ. Aliquots of 5×10⁵ cells were centrifuged for 3 min at300 g and resuspended in 50 μl of either 1 μM BDB or cp33 in thepresence or absence of a control monoclonal human IgG1 (Nip228,Medlmmune Ltd) at concentrations in the range 0.001-5 μM. The stainingand FACS analysis were performed as described above.

FIG. 5 demonstrates that as the concentration of human IgG1 increasesabove approximated 10 nM, both the BDB and cp33 peptide are displacedfrom binding to IFGγ-activated U937 cells. This competition isconsistent with the BDB and cp33 peptides binding to the Fc receptor.

Example 6: Demonstrating the Colocalization of the BDB and Amyloid tothe Surface of U937 Cells

IFNγ-activated U937 cells, a human macrophage cell line, were incubatedwith either BDB, cp33 peptide, or vehicle in the presence or absence ofAlexa Fluor™ 647-labelled hLys amyloid in order to determine if theIFNγ-activated U937 cells have increased binding of amyloid in thepresence of BDB or cp33 peptide as compared to vehicle.

Specifically, hLys were labelled with the fluorescent dye alexa 647,using the Alexa Fluor™ 647 Microscale Protein Labelling Kit (ThermoFisher Scientific) according to the manufacturer's instructions with thefollowing modifications: after protein labelling, the solution was madeup to 500 μl with PBS and the sample centrifuged at 20800 for 40 min.The supernatant was discarded and the pellet washed with PBS,resuspended in 500 μl PBS and the centrifugation repeated. The finalpellet was resuspended in 100 μl of PBS.

U937 cells were cultured in RPMI 1640+2 mM Glutamine+10% v/v FCS for 48h in the presence of 100 μg/ml human IFNγ. Aliquots of 5×10⁵ cells werecentrifuged 3 min at 300 g and resuspended in 50 μl of either 1 μM BDBor cp33 and then incubated with either 1 or 0.1 μM AF647-hLyz. Thestaining and FACS analysis were performed as before, this time reportingthe percentage of live cells positive for both AF488-streptavidin andAF647 h-Lyz.

FIG. 6 compares the localization of amyloid to the surface ofIFNγ-activated U937 cells treated with BDB, cp33 peptide, or vehiclecontrol. Both BDB and cp33 peptide show cell binding irrespective ofwhether amyloid is present (displacement on y-axis in top four panels).Up to 14.4% of IFNγ-activated U937 cells are observed binding amyloid inthe presence of BDB as compared to 1.0% in the presence of cp33 peptideor 0.4% when treated with vehicle. These results demonstrate that BDBenhances the binding of amyloid to IFGγ-activated U937 cells.

Example 7: Measuring Phagocytosis of Labelled Amyloid

Macrophage-differentiated U937 cells were co-incubated with pHrodo-redlabelled amyloid, BDB, cp33 peptide, or control in order to determinethe amount of amyloid phagocytosed under various conditions. hLysamyloid fibrils were generated as described above and were labelled withthe pHrodo Red Microscale Labeling Kit (ThermoFisher) according to themanufacturers instruction except that unincorporated label was removedfrom the labelled amyloid by three rounds of centrifuging at 20,000×g ina microfuge, discarding the supernatant and resuspending the amyloidpellet in PBS. U937 cells were differentiated into macrophages in thepresence of 100 μg/ml M-CSF (Peprotech), 1 μg/ml PMA (Sigma) and 100μg/ml IFNγ (Thermo Fisher Scientific) for 4 days. 8×10⁵ cells/conditionwere centrifuged for 3 min at 300 g and resuspended in 50 μl of 1 μMBDB, 1 μM cp33, 1 μM BDB with 0.1 μM labelled amyloid, 1 μM cp33 with0.1 μM labelled amyloid, 0.1 μM labelled amyloid or a 1:100 PBS dilutionof a pHrodo Red-labelled E. coli suspension (Thermo Fisher Scientific).The cells were incubated at 4° C. for 1 h, centrifuged for 3 min at 100g and resuspended in 300 μl of RPMI media containing 100 μg/ml M-CSF, 1μg/ml PMA and 100 μg/ml IFNg. For each condition, a 100 μl suspension ofcells was plated to the wells of black 96-well plates with clear bottom(Greiner Bio-one).

The plates were placed in an IncuCyte instrument and settings programmedto scan both in phase contrast and the red fluorescence channel hourlyfor 48 h. Raw data for each time point was then processed using theIncuCyte Zoom software with a red channel processing algorithm. A metricof Total Red Object Area (μm²/well) was used to quantify the appearanceof pHrodo red fluorescence within cells.

FIG. 7 demonstrates that BDB enhances the phagocytosis of amyloid bymacrophage-differentiated U937 cells. Red colour (y-axis) reportedphagocytosed amyloid; this signal increased more rapidly and achieved agreater amplitude in cells treated with BDB-amyloid (1) as compared tocp33-amyloid (3) or amyloid alone (2). pHrodo-red E. coli were added asa positive control (4). In the absence of amyloid, BDB (5) and the cp33peptide (6) did not generate a signal.

In a separate experiment, several different BDB constructs were eachtested in the same phagocytosis assay discussed above. In thisexperiment, the BDB constructs comprised the structure of Compound III:

wherein R¹ was biotin (which may be optionally replaced with hydrogen);R² was:

a) the dye alone

b) the dye with a beta-alanine linker

c) the dye with a 6-amino-4-oxahexanoate linker

d) the dye with a 9-amino-4,7-dioxanonanoate linker

In this experiment, it was surprisingly found that the BDB with the morerigid beta-alanine linker induced a more than two-fold greater rate ofamyloid phagocytosis as compared to the BDB lacking the linker or to theBDBs having the longer 6-amino-4-oxahexanoate or9-amino-4,7-dioxanonanoate linkers (FIG. 8).

Example 8: Structural Effects of Adding Linkers Between the AmyloidBinding and FcR-Binding Moieties

Space-filling models were developed to determine the structural effectsof adding linkers between the amyloid binding and FcR-binding moieties.Specifically, FIG. 9 demonstrates a comparison between models which useno linker, a β-Ala linker, a short PEG linker, and a long PEG linker(FMOC derivatized β-Alanine; 9-amino-4,7-dioxanonanoic acid; and6-amino-4-oxaohexanoic acid).

We claim:
 1. A conjugate comprising a peptide having the amino acidsequence of AQVNSCLLLPNLLGCGDDK (SEQ ID NO: 3) fused to a targetingmoiety that targets molecular aggregates having the formula of CompoundI:


2. A method of treating an amyloidosis by administering any one or moreof the conjugates of claim
 1. 3. The method of claim 2, wherein theamyloidosis is selected from the group consisting of: AL amyloidosis, AAamyloidosis, Alzheimer Disease, LECT2 amyloidosis, leptomeningealamyloidosis, senile systemic amyloidosis, familial amyloidpolyneuropathy, haemodialysis-associated amyloidosis, type 2 diabetes,Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,Finnish type amyloidosis, cerebral amyloid angiopathy, familial visceralamyloidosis, primary cutaneous amyloidosis prolactinoma, familialcorneal amyloidosis, Parkinson's Disease, amyotrophic lateral sclerosis,cerebral amyloid angiopathy, frontotemporal lobar dementia, medullarythyroid carcinoma, and B2M amyloidosis.
 4. The method of claim 2,wherein the amyloidosis is a familial amyloidosis.
 5. The method ofclaim 4, wherein the amyloidosis is systemic amyloidosis.
 6. A method ofreducing levels of an aggregate in a cell or tissue by treating the cellor tissue with the conjugate of claim
 1. 7. A conjugate comprising thestructure of Compound III:

wherein R¹ is biotin or hydrogen; wherein R² comprises:


8. A method of treating an amyloidosis by administering any one or moreof the conjugates of claim
 7. 9. The method of claim 8, wherein theamyloidosis is selected from the group consisting of: AL amyloidosis, AAamyloidosis, Alzheimer Disease, LECT2 amyloidosis, leptomeningealamyloidosis, senile systemic amyloidosis, familial amyloidpolyneuropathy, haemodialysis-associated amyloidosis, type 2 diabetes,Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,Finnish type amyloidosis, cerebral amyloid angiopathy, familial visceralamyloidosis, primary cutaneous amyloidosis prolactinoma, familialcorneal amyloidosis, Parkinson's Disease, amyotrophic lateral sclerosis,cerebral amyloid angiopathy, frontotemporal lobar dementia, medullarythyroid carcinoma, and B2M amyloidosis.
 10. The method of claim 8,wherein the amyloidosis is a familial amyloidosis.
 11. The method ofclaim 10, wherein the amyloidosis is systemic amyloidosis.
 12. A methodof reducing levels of an aggregate in a cell or tissue by treating thecell or tissue with the conjugate of claim 7.